A fundamental rule of molecular biology, with few exceptions, is the universality of the genetic code. While certain among degenerate codons may be favored by one type of organism or another, the same sequence of codons most usually produces the same sequence of amino acids regardless of whether the cell effecting expression is an E. coli, a yeast, or a cell derived from a human being or a petunia. Such versatility is not extended to the control sequences which effect the expression of the coding portions of the DNA. Promoters which are operable in bacteria do not, as a general rule, operate in eucaryotic hosts. There are, of course, some exceptions related to promoters associated with virulence of certain bacteria to their targeted host organisms. There are also other fortuitous exceptions wherein bacterial promoters operate, though more poorly, in yeast. Similarly, control sequences which are normally utilized by mammalian cells do not operate in unicellular eucaryotic hosts such as yeast or in procaryotes such as bacteria. Thus, the standard approach to securing the production of desired peptide in a particular host is to ligate the coding sequence for the desired peptide in suitable juxtaposition to control sequences indigenous to the host used to produce the protein.
In eucaryotic sytems, at least two and in some cases three, elements of control sequences are considered relevant to successful expression: a sequence 5' of the start codon which is responsible for the initiation of transcription (the promoter) and a sequence 3' of the cdding sequence which appears to contain at least a polyadenylation signal which apparently is instrumental in transporting the RNA transcript from the nucleus to the cytoplasm (the terminator). Thus, to secure expression in a eucaryote, it has been necessary to provide indigenous promoters and terminators in operable linkage to the desired coding sequence.
In addition, it has recently been shown that "enhancers" of expression may be involved in protein production for certain specialized mammalian cells. Enhancers of viral origin which can operate in eucaryotic hosts have been known for some time. These enhancer sequences are apparently relatively insensitive to orientation and position, and function to increase expression levels of associated expression packages.
Yeast hosts have been successfully transformed and induced to produce protein sequences using a variety of yeast origin vectors containing yeast promoters and terminators. See, for example, Broach, J. R., Meth. Enz (1983) 101:307; Stinchcomb, et al, Nature (1979) 282: 39, Tschempe, et al, Gene (1980) 10:157 and Clark, L., et al, Meth. Enz (1983) 101:300, Holland, M. J., et al, J Biol Chem (1981) 256:1385. The variety of control sequences available is quite large, and includes promoters for the synthesis of the glycolytic enzymes, for alcohol dehydrogenase, acid phosphatase, and a variety of others.
Because yeast are capable of rapid and luxuriant growth under aerobic conditions, they are ideal candidates for large scale production of proteins. Also, by altering their complement of enzymic catalysts, they may be employed to carry out chemical transformations such as hydroxylation, oxidation, isomerization, hydrolysis or utilization of targeted chemicals. Accordingly, the provision of suitable control sequences in yeast hosts provides a useful method of employing these organisms for the production of proteins and other materials. It has, heretofore, been necessary to employ control sequences of yeast origin in order to do this.
Known yeast control sequences can provide useful results; however, they have certain associated drawbacks. Since they are essentially endogenous, efforts to control heterologous protein production through control of the operably linked control sequences may have the side effect of causing undesired fluctuations in expression of the analogous endogenous system. Also, sequence identity with native controls may result in unwanted recombination into the host DNA. Finally, expression vectors intended for other species hosts, e.g., mammalian cells, which are more difficult to culture than yeast, cannot use yeast as a convenient cloning and expression manipulation host.
(In connection with the last-mentioned problem, it should be noted that versatility with respect to host, if extended to procaryotes as well, would constitute an even greater advantage for an expression control system. Control sequences which are operable in bacteria, yeast and mammalian cells offer, for example, the opportunity to study expression under a wide range of post translational processing conditions.)
In short, the necessity to use yeast control sequences carries an intrinsic limitation to the characteristics to these particular control sequences. To provide greater flexibility and control of expression in yeast, it would be desirable to add xenogeneic control sequences to the available repetoire. The present invention provides for such an increase in versatility.